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Fig. 5. Ciliary targeting of the Inv C-terminus depends on the IQ2 domain. (A) Localization of calmodulin was observed by immunocytochemistry. Calmodulin was detected by the anti-calmodulin antibody (green) and the primary cilium was detected by anti-acetylated -tubulin antibody (ac-tubulin; red). The calmodulin signal is predominantly present at the peri-basal-body area. Calmodulin staining in primary cilia is faint and distributed over the entire length of the primary cilium. Calmodulin localization in primary cilia and the peri-basal body is identical in wild-type cells and inv cells. Scale bars: 10 µm. (B) The IQ2 domain of mouse Inv is aligned with human and rat Inv. The construct in which glutamic acid (E) is substituted for isoleucine (I) in Inv (742-1062: I921E) is indicated in red. (C) GFP-tagged Inv (742-1062) and Inv (742-1062: I921E) proteins were expressed and ciliary targeting was studied. Mutation of an amino acid in the IQ2 domain resulted in loss of ciliary targeting. Arrowheads indicate GFP signal and primary cilia. Percentages of ciliated cells with positive ciliary GFP signal in transfected cells are indicated on the right. (D) Detection of direct calmodulin binding to the IQ2 domain. GST-fused truncated-Inv proteins expressed in E. coli under IPTG induction are shown. Inv (742-1062) binds to calmodulin, but deletion or mutation of the IQ2 domain results in loss of calmodulin binding (upper panel; arrowhead). Expression of GST fusion proteins was confirmed by anti-GST antibody staining (lower panel).

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